Strategies for high throughput identification and detection of polymorphisms

ABSTRACT

The invention relates to a method for identifying one or more polymorphisms in nucleic acid samples, comprising: (a) performing a reproducible complexity reduction on a plurality of nucleic acid samples to provide a plurality of libraries of the nucleic acid samples comprising amplified fragments, wherein the reproducible complexity reduction comprises amplifying fragments of the nucleic acid samples using one or more primers to obtain the amplified fragments, and wherein the amplified fragments in each library comprise a unique identifier sequence to indicate origin of each library obtained by the reproducible complexity reduction; (b) combining the plurality of libraries to obtain a combined library and sequencing at least a portion of the combined library to obtain sequences; (c) aligning the sequences to obtain an alignment; and (d) identifying one or more polymorphisms in the plurality of nucleic acid samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application of U.S. patentapplication Ser. No. 15/350,441, filed Nov. 14, 2016, which is aContinuation application of U.S. patent application Ser. No. 14/626,822,filed Feb. 19, 2015, which is a Continuation application of U.S. patentapplication Ser. No. 14/253,806, now U.S. Pat. No. 9,023,768, which is aContinuation application of U.S. patent application Ser. No. 11/993,945,now U.S. Pat. No. 8,785,353, which is a National Stage entry ofInternational Patent Application No. PCT/NL2006/000311, filed Jun. 23,2006, which claims benefit under 35 U.S.C. §119(e) of U.S. ProvisionalApplication Nos. 60/693,053, filed Jun. 23, 2005 and 60/759,034, filedJan. 17, 2006 and which also claims priority to European Application No.06075104.7, filed Jan. 16, 2006. Each of these applications is herebyincorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 22, 2017, isnamed 085342-1302SequenceListing.txt and is 17.2 KB in size.

TECHNICAL FIELD

The present invention relates to the fields of molecular biology andgenetics. The invention relates to rapid identification of multiplepolymorphisms in a nucleic acid sample. The identified polymorphisms maybe used for development of high-throughput screening systems forpolymorphisms in test samples.

BACKGROUND OF THE INVENTION

Exploration of genomic DNA has long been desired by the scientific, inparticular medical, community. Genomic DNA holds the key toidentification, diagnosis and treatment of diseases such as cancer andAlzheimer's disease. In addition to disease identification andtreatment, exploration of genomic DNA may provide significant advantagesin plant and animal breeding efforts, which may provide answers to foodand nutrition problems in the world.

Many diseases are known to be associated with specific geneticcomponents, in particular with polymorphisms in specific genes. Theidentification of polymorphisms in large samples such as genomes is atpresent a laborious and time-consuming task. However, suchidentification is of great value to areas such as biomedical research,developing pharmacy products, tissue typing, genotyping and populationstudies.

SUMMARY OF THE INVENTION

The present invention provides for a method of efficiently identifyingand reliably detecting polymorphisms in a complex, e.g. very large,nucleic acid sample (e.g. DNA or RNA) in a rapid and economical mannerusing a combination of high-throughput methods.

This integration of high-throughput methods together provide a platformthat is particularly suited for the rapid and reliable identificationand detection of polymorphisms in highly complex nucleic acid sampleswherein conventional identification and mapping of polymorphisms wouldbe laborious and time-consuming.

One of the things the present inventors have found is a solution for theidentification of polymorphisms, preferably Single NucleotidePolymorphisms, but likewise for (micro)satellites and/or indels, inparticular in large genomes. The method is unique in its applicabilityto large and small genomes alike, but provides particular advantages forlarge genomes, in particular polyploidal species.

To identify SNPs (and subsequently detect the identified SNPs) there areseveral possibilities available in the art. In a first option the wholegenome can be sequenced, and this can be done so for severalindividuals. This is mostly a theoretical exercise as this is cumbersomeand expensive and, despite the rapid development of technology simplynot feasible to do for every organism, especially the ones with largergenomes. Second option is to use available (fragmented) sequenceinformation, such as EST libraries. This allows the generation of PCRprimers, resequencing and comparison between individuals. Again, thisrequires initial sequence information that is not available or only in alimited amount. Furthermore separate PCR-assays have to be developed foreach region which adds enormously to costs and development time.

The third option is to limit one self to part of the genome for eachindividual. The difficulty resides that the provided part of the genomemust be the same for different individuals in order to providecomparable result for successful SNP identification. The presentinventors now have solved this dilemma by integration of highlyreproducible methods for selecting part of genome with high throughputsequencing for the identification of polymorphisms integrated withsample preparation and high throughput identification platforms. Thepresent invention accelerates the process of polymorphism discovery anduses the same elements in the subsequent process for the exploitation ofthe discovered polymorphisms to allow for effective and reliable highthroughput genotyping.

Further envisaged applications of the method of the present inventioninclude screening enriched microsatellite libraries, performingtranscript profiling cDNA-AFLP (digital Northern), sequencing of complexgenomes, EST library sequencing (on whole cDNA or cDNA-AFLP), microRNAdiscovery (sequencing of small insert libraries), Bacterial ArtificialChromosome (BAC) (contig) sequencing, Bulked Segregant analysis approachAFLP/cDNA-AFLP, routine detection of AFLP fragments, e.g. formarker-assisted back-crosses (MABC), etcetera.

Definitions

In the following description and examples a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided. Unless otherwise defined herein,all technical and scientific terms used have the same meaning ascommonly understood by one of ordinary skill in the art to which thisinvention belongs. The disclosures of all publications, patentapplications, patents and other references are incorporated herein intheir entirety by reference.

Polymorphism: polymorphism refers to the presence of two or morevariants of a nucleotide sequence in a population. A polymorphism maycomprise one or more base changes, an insertion, a repeat, or adeletion. A polymorphism includes e.g. a simple sequence repeat (SSR)and a single nucleotide polymorphism (SNP), which is a variation,occurring when a single nucleotide: adenine (A), thymine (T), cytosine(C) or guanine (G)—is altered. A variation must generally occur in atleast 1% of the population to be considered a SNP. SNPs make up e.g. 90%of all human genetic variations, and occur every 100 to 300 bases alongthe human genome. Two of every three SNPs substitute Cytosine (C) withThymine (T). Variations in the DNA sequences of e.g. humans or plantscan affect how they handle diseases, bacteria, viruses, chemicals,drugs, etc.

Nucleic acid: a nucleic acid according to the present invention mayinclude any polymer or oligomer of pyrimidine and purine bases,preferably cytosine, thymine, and uracil, and adenine and guanine,respectively (See Albert L. Lehninger, Principles of Biochemistry, at793-800 (Worth Pub. 1982) which is herein incorporated by reference inits entirety for all purposes). The present invention contemplates anydeoxyribonucleotide, ribonucleotide or peptide nucleic acid component,and any chemical variants thereof, such as methylated, hydroxymethylatedor glycosylated forms of these bases, and the like. The polymers oroligomers may be heterogenous or homogenous in composition, and may beisolated from naturally occurring sources or may be artificially orsynthetically produced. In addition, the nucleic acids may be DNA orRNA, or a mixture thereof, and may exist permanently or transitionallyin single-stranded or double-stranded form, including homoduplex,heteroduplex, and hybrid states.

Complexity reduction: the term complexity reduction is used to denote amethod wherein the complexity of a nucleic acid sample, such as genomicDNA, is reduced by the generation of a subset of the sample. This subsetcan be representative for the whole (i.e. complex) sample and ispreferably a reproducible subset. Reproducible means in this contextthat when the same sample is reduced in complexity using the samemethod, the same, or at least comparable, subset is obtained. The methodused for complexity reduction may be any method for complexity reductionknown in the art. Examples of methods for complexity reduction includefor example AFLP® (Keygene N.V., the Netherlands; see e.g. EP 0 534858), the methods described by Dong (see e.g. WO 03/012118, WO00/24939), indexed linking (Unrau et al., vide infra), etc. Thecomplexity reduction methods used in the present invention have incommon that they are reproducible. Reproducible in the sense that whenthe same sample is reduced in complexity in the same manner, the samesubset of the sample is obtained, as opposed to more random complexityreduction such as microdissection or the use of mRNA (cDNA) whichrepresents a portion of the genome transcribed in a selected tissue andfor its reproducibilty is depending on the selection of tissue, time ofisolation etc.

Tagging: the term tagging refers to the addition of a tag to a nucleicacid sample in order to be able to distinguish it from a second orfurther nucleic acid sample. Tagging can e.g. be performed by theaddition of a sequence identifier during complexity reduction or by anyother means known in the art. Such sequence identifier can e.g. be aunique base sequence of varying but defined length uniquely used foridentifying a specific nucleic acid sample. Typical examples thereof arefor instance ZIP sequences. Using such tag, the origin of a sample canbe determined upon further processing. In case of combining processedproducts originating from different nucleic acid samples, the differentnucleic acid samples should be identified using different tags.

Tagged library: the term tagged library refers to a library of taggednucleic acid.

Sequencing: The term sequencing refers to determining the order ofnucleotides (base sequences) in a nucleic acid sample, e.g. DNA or RNA.

Aligning and alignment: With the term “aligning” and “alignment” ismeant the comparison of two or more nucleotide sequence based on thepresence of short or long stretches of identical or similar nucleotides.Several methods for alignment of nucleotide sequences are known in theart, as will be further explained below.

Detection probes: The term “detection probes” is used to denote probesdesigned for detecting a specific nucleotide sequence, in particularsequences containing one or more polymorphisms.

High-throughput screening: High-throughput screening, often abbreviatedas HTS, is a method for scientific experimentation especially relevantto the fields of biology and chemistry. Through a combination of modernrobotics and other specialised laboratory hardware, it allows aresearcher to effectively screen large amounts of samplessimultaneously.

Test sample nucleic acid: The term “test sample nucleic acid” is used toindicate a nucleic acid sample that is investigated for polymorphismsusing the method of the present invention.

Restriction endonuclease: a restriction endonuclease or restrictionenzyme is an enzyme that recognizes a specific nucleotide sequence(target site) in a double-stranded DNA molecule, and will cleave bothstrands of the DNA molecule at every target site.

Restriction fragments: the DNA molecules produced by digestion with arestriction endonuclease are referred to as restriction fragments. Anygiven genome (or nucleic acid, regardless of its origin) will bedigested by a particular restriction endonuclease into a discrete set ofrestriction fragments. The DNA fragments that result from restrictionendonuclease cleavage can be further used in a variety of techniques andcan for instance be detected by gel electrophoresis.

Gel electrophoresis: in order to detect restriction fragments, ananalytical method for fractionating double-stranded DNA molecules on thebasis of size can be required. The most commonly used technique forachieving such fractionation is (capillary) gel electrophoresis. Therate at which DNA fragments move in such gels depends on their molecularweight; thus, the distances travelled decrease as the fragment lengthsincrease. The DNA fragments fractionated by gel electrophoresis can bevisualized directly by a staining procedure e.g. silver staining orstaining using ethidium bromide, if the number of fragments included inthe pattern is sufficiently small. Alternatively further treatment ofthe DNA fragments may incorporate detectable labels in the fragments,such as fluorophores or radioactive labels.

Ligation: the enzymatic reaction catalyzed by a ligase enzyme in whichtwo double-stranded DNA molecules are covalently joined together isreferred to as ligation. In general, both DNA strands are covalentlyjoined together, but it is also possible to prevent the ligation of oneof the two strands through chemical or enzymatic modification of one ofthe ends of the strands. In that case the covalent joining will occur inonly one of the two DNA strands.

Synthetic oligonucleotide: single-stranded DNA molecules havingpreferably from about 10 to about 50 bases, which can be synthesizedchemically are referred to as synthetic oligonucleotides. In general,these synthetic DNA molecules are designed to have a unique or desirednucleotide sequence, although it is possible to synthesize families ofmolecules having related sequences and which have different nucleotidecompositions at specific positions within the nucleotide sequence. Theterm synthetic oligonucleotide will be used to refer to DNA moleculeshaving a designed or desired nucleotide sequence.

Adaptors: short double-stranded DNA molecules with a limited number ofbase pairs, e.g. about 10 to about 30 base pairs in length, which aredesigned such that they can be ligated to the ends of restrictionfragments. Adaptors are generally composed of two syntheticoligonucleotides which have nucleotide sequences which are partiallycomplementary to each other. When mixing the two syntheticoligonucleotides in solution under appropriate conditions, they willanneal to each other forming a double-stranded structure. Afterannealing, one end of the adaptor molecule is designed such that it iscompatible with the end of a restriction fragment and can be ligatedthereto; the other end of the adaptor can be designed so that it cannotbe ligated, but this need not be the case (double ligated adaptors).

Adaptor-ligated restriction fragments: restriction fragments that havebeen capped by adaptors.

Primers: in general, the term primers refers to a DNA strand which canprime the synthesis of DNA. DNA polymerase cannot synthesize DNA de novowithout primers: it can only extend an existing DNA strand in a reactionin which the complementary strand is used as a template to direct theorder of nucleotides to be assembled. We will refer to the syntheticoligonucleotide molecules which are used in a polymerase chain reaction(PCR) as primers.

DNA amplification: the term DNA amplification will be typically used todenote the in vitro synthesis of double-stranded DNA molecules usingPCR. It is noted that other amplification methods exist and they may beused in the present invention without departing from the gist.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for a method for identifying one or morepolymorphisms, said method comprising the steps of:

-   -   a) providing a first nucleic acid sample of interest;    -   b) performing a complexity reduction on the first nucleic acid        sample of interest to provide a first library of the first        nucleic acid sample;    -   c) consecutively or simultaneously performing steps a) and b)        with a second or further nucleic acid sample of interest to        obtain a second or further library of the second or further        nucleic acid sample of interest;    -   d) sequencing of at least a portion of the first library and the        second or further library;    -   e) aligning the sequences obtained in step d);    -   f) determining one or more polymorphisms between the first        nucleic acid sample and second or further nucleic acid sample in        the alignment of step e);    -   g) using the one or more polymorphisms determined in step f) to        design one or more detection probes;    -   h) providing a test sample nucleic acid of interest;    -   i) performing the complexity reduction of step b) on the test        sample nucleic acid of interest to provide a test library of the        test sample nucleic acid;    -   j) subjecting the test library to high-throughput screening to        identify the presence, absence or amount of the polymorphisms        determined in step f) using the one or more detection probes        designed in step g).

In step a), a first nucleic acid sample of interest is provided. Saidfirst nucleic acid sample of interest is preferably a complex nucleicacid sample such as total genomic DNA or a cDNA library. It is preferredthat the complex nucleic acid sample is total genomic DNA.

In step b), a complexity reduction is performed on the first nucleicacid sample of interest to provide a first library of the first nucleicacid sample.

In one embodiment of the invention, the step of complexity reduction ofthe nucleic acid sample comprises enzymatically cutting the nucleic acidsample in restriction fragments, separating the restriction fragmentsand selecting a particular pool of restriction fragments. Optionally,the selected fragments are then ligated to adaptor sequences containingPCR primer templates/binding sequences.

In one embodiment of complexity reduction, a type IIs endonuclease isused to digest the nucleic acid sample and the restriction fragments areselectively ligated to adaptor sequences. The adaptor sequences cancontain various nucleotides in the overhang that is to be ligated andonly the adaptor with the matching set of nucleotides in the overhang isligated to the fragment and subsequently amplified. This technology isdepicted in the art as ‘indexing linkers’. Examples of this principlecan be found inter alia in Unrau P. and Deugau K. V. (1994) Gene145:163-169.

In another embodiment, the method of complexity reduction utilizes tworestriction endonucleases having different target sites and frequenciesand two different adaptor sequences.

In another embodiment of the invention, the step of complexity reductioncomprises performing an Arbitrarily Primed PCR upon the sample.

In yet another embodiment of the invention, the step of complexityreduction comprises removing repeated sequences by denaturing andreannealing the DNA and then removing double-stranded duplexes.

In another embodiment of the invention, the step of complexity reductioncomprises hybridizing the nucleic acid sample to a magnetic bead whichis bound to an oligonucleotide probe containing a desired sequence. Thisembodiment may further comprise exposing the hybridized sample to asingle strand DNA nuclease to remove the single-stranded DNA, ligatingan adaptor sequence containing a Class IIs restriction enzyme to releasethe magnetic bead. This embodiment may or may not comprise amplificationof the isolated DNA sequence. Furthermore, the adaptor sequence may ormay not be used as a template for the PCR oligonucleotide primer. Inthis embodiment, the adaptor sequence may or may not contain a sequenceidentifier or tag.

In another embodiment, the method of complexity reduction comprisesexposing the DNA sample to a mismatch binding protein and digesting thesample with a 3′ to 5′ exonuclease and then a single strand nuclease.This embodiment may or may not include the use of a magnetic beadattached to the mismatch binding protein.

In another embodiment of the present invention, complexity reductioncomprises the CHIP method as described herein elsewhere or the design ofPCR primers directed against conserved motifs such as SSRs, NBS regions(nucleotide biding regions), promoter/enhancer sequences, telomerconsensus sequences, MADS box genes, ATP-ase gene families and othergene families.

In step c), steps a) and b) are consecutively or simultaneouslyperformed with a second or further nucleic acid sample of interest toobtain a second or further library of the second or further nucleic acidsample of interest. Said second or further nucleic acid sample ofinterest is preferably also a complex nucleic acid sample such as totalgenomic DNA. It is preferred that the complex nucleic acid sample istotal genomic DNA. It is also preferred that said second or furthernucleic acid sample is related to the first nucleic acid sample. Thefirst nucleic acid sample and the second or further nucleic acid may forexample be different lines of a plant, such as different pepper lines,or different varieties. Steps a) en b) may be performed for merely asecond nucleic acid sample of interest, but may also additionally beperformed for a third, fourth, fifth, etc. nucleic acid sample ofinterest.

It is to be noted that the method according to the present inventionwill be most useful when complexity reduction is performed using thesame method and under substantially the same, preferably identical,conditions for the first nucleic acid sample and the second or furthernucleic acid sample. Under such conditions, similar (comparable)fractions of the (complex) nucleic acid samples will be obtained.

In step d), at least a portion of the first library and of the second orfurther library is sequenced. It is preferred that the amount of overlapof sequenced fragments from the first library and second or furtherlibrary is at least 50%, more preferably at least 60%, yet morepreferably at least 70%, even more preferably at least 80%, yet morepreferably at least 90%, and most preferably at least 95%.

The sequencing may in principle be conducted by any means known in theart, such as the dideoxy chain termination method. It is howeverpreferred that the sequencing is performed using high-throughputsequencing methods, such as the methods disclosed in WO 03/004690, WO03/054142, WO 2004/069849, WO 2004/070005, WO 2004/070007, and WO2005/003375 (all in the name of 454 Corporation), by Seo et al. (2004)Proc. Natl. Acad. Sci. USA 101:5488-93, and technologies of Helios,Solexa, US Genomics, etcetera, which are herein incorporated byreference. It is most preferred that sequencing is performed using theapparatus and/or method disclosed in WO 03/004690, WO 03/054142, WO2004/069849, WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all inthe name of 454 Corporation), which are herein incorporated byreference. The technology described allows sequencing of 40 millionbases in a single run and is 100 times faster and cheaper than competingtechnology. The sequencing technology roughly consists of 4 steps: 1)fragmentation of DNA and ligation of specific adaptor to a library ofsingle-stranded DNA (ssDNA); 2) annealing of ssDNA to beads andemulsification of the beads in water-in-oil microreactors; 3) depositionof DNA carrying beads in a PicoTiterPlate®; and 4) simultaneoussequencing in 100,000 wells by generation of a pyrophosphate lightsignal. The method will be explained in more detail below.

In step e), the sequences obtained in step d) are aligned to provide analignment. Methods of alignment of sequences for comparison purposes arewell known in the art. Various programs and alignment algorithms aredescribed in: Smith and Waterman (1981) Adv. Appl. Math. 2:482;Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman(1988) Proc. Natl. Acad. Sci. USA 85:2444; Higgins and Sharp (1988) Gene73:237-244; Higgins and Sharp (1989) CABIOS 5:151-153; Corpet et al.(1988) Nucl. Acids Res. 16:10881-90; Huang et al. (1992) Computer Appl.in the Biosci. 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol.24:307-31, which are herein incorporated by reference. Altschul et al.(1994) Nature Genet. 6:119-29 (herein incorporated by reference) presenta detailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al.,1990) is available from several sources, including the National Centerfor Biological Information (NCBI, Bethesda, Md.) and on the Internet,for use in connection with the sequence analysis programs blastp,blastn, blastx, tblastn and tblastx. A further application can be inmicrosatellite mining (see Varshney et al. (2005) Trends in Biotechn.23(1):48-55.

Typically, the alignment is performed on the sequence data that havebeen trimmed for the adaptors/primer and/or identifiers, i.e. using onlythe sequence data from the fragments that originate from the nucleicacid sample. Typically, the sequence data obtained are used foridentifying the origin of the fragment (i.e. from which sample), thesequences derived from the adaptor and/or identifier are removed fromthe data and alignment is performed on this trimmed set.

In step f), one or more polymorphisms are determined between the firstnucleic acid sample and second or further nucleic acid sample in thealignment. The alignment can be made such that the sequences derivedfrom the first nucleic acid sample and the second or further nucleicacid sample can be compared. Differences can then be identifiedreflecting polymorphisms.

In step g) the one or more polymorphisms determined in step g) are usedto design detection probes, for example for detection by hybridizationon DNA chips or a beads-based detection platform. The detection probesare designed such that a polymorphism is reflected therein. In case ofsingle nucleotide polymorphisms (SNPs) the detection probes typicallycontain the variant SNP alleles at the central position such as tomaximize allele discrimination. Such probes can advantageously be usedto screen test samples having a certain polymorphism. The probes can besynthesized using any method known in the art. The probes are typicallydesigned such that they are suitable for high throughput screeningmethods.

In step h), a test sample nucleic acid of interest is provided. The testsample nucleic acid may be any sample, but is preferably another line orvariety to be mapped for polymorphisms. Commonly, a collection of testsamples representing the germplasm of the organisms studied is used tovalidate experimentally that the (SN) polymorphism is genuine anddetectable and to calculate allele frequencies of the observed alleles.Optionally, samples of a genetic mapping population are included in thevalidation step in order to determine the genetic map position of thepolymorphism too.

In step i), the complexity reduction of step b) is performed on the testsample nucleic acid of interest to provide a test library of the testsample nucleic acid. It is highly preferred that throughout the methodaccording to the present invention the same method for complexityreduction is used using substantially the same, preferably identical,conditions, thus covering a similar fraction of the sample. It ishowever not required that a tagged test library is obtained, although atag may be present on the fragments in the test library.

In step j) the test library is subjected to high-throughput screening toidentify the presence, absence or amount of the polymorphisms determinedin step f) using the detection probes designed in step g). One skilledin the art knows several methods for high-throughput screening usingprobes. It is preferred that one or more probes designed using theinformation obtained in step g) are immobilized on an array, such as aDNA chip, and that such array is subsequently contacted with the testlibrary under hybridizing conditions. DNA fragments within the testlibrary complementary to one or more probes on the array will under suchconditions hybridize to such probes, and can thus be detected. Othermethods of high-throughput screening are also envisaged within the scopeof the present invention, such as immobilization of the test libraryobtained in step j) and contacting of said immobilized test library withthe probes designed in step h) under hybridizing conditions.

Another high-throughput sequencing screening technique is provided interalia by Affymetrix using chip-based detection of SNPs and beadtechnology provided by Illumina.

In an advantageous embodiment, step b) in the method according to thepresent invention further comprises the step of tagging of the libraryto obtain a tagged library, and said method further comprising step c1)of combining the first tagged library and second or further taggedlibrary to obtain a combined library.

It is preferred that the tagging is performed during the complexityreduction step as to reduce the amount of steps required to obtain thefirst tagged library of the first nucleic acid sample. Such simultaneoustagging can e.g. be achieved by AFLP, using adaptors that comprise aunique (nucleotide) identifier for each sample.

The tagging is intended to distinguish between samples of differentorigin, e.g. obtained from different plant lines, when the libraries oftwo or more nucleic acid samples are combined to obtain a combinationlibrary. Thus, preferably different tags are used for preparing thetagged libraries of the first nucleic acid sample and the second orfurther nucleic acid sample. When for example five nucleic acid samplesare used, it is intended to obtain five differently tagged libraries,the five different tags denoting the respective original samples.

The tag may be any tag known in the art for distinguishing nucleic acidsamples, but is preferably a short identifier sequence. Such identifiersequence can e.g. be a unique base sequence of varying length used toindicate the origin of the library obtained by complexity reduction.

In a preferred embodiment, the tagging of the first library and thesecond or further library is performed using different tags. Asdiscussed above, it is preferred that each library of a nucleic acidsample is identified by its own tag. The test sample nucleic acid doesnot need to be tagged.

In a preferred embodiment of the invention, the complexity reduction isperformed by means of AFLP® (Keygene N. V., the Netherlands; see e.g. EP0 534 858 and Vos et al. (1995). AFLP: a new technique for DNAfingerprinting, Nucleic Acids Research, vol. 23, no. 21, 4407-4414,which are herein incorporated in their entirety by reference).

AFLP is a method for selective restriction fragment amplification. AFLPdoes not any prior sequence information and can be performed on anystarting DNA. In general, AFLP comprises the steps of:

-   -   (a) digesting a nucleic acid, in particular a DNA or cDNA, with        one or more specific restriction endonucleases, to fragment the        DNA into a corresponding series of restriction fragments;    -   (b) ligating the restriction fragments thus obtained with a        double-stranded synthetic oligonucleotide adaptor, one end of        which is compatible with one or both of the ends of the        restriction fragments, to thereby produce adapter-ligated,        preferably tagged, restriction fragments of the starting DNA;    -   (c) contacting the adapter-ligated, preferably tagged,        restriction fragments under hybridizing conditions with at least        one oligonucleotide primer that contains at least one selective        nucleotide at its 3′-end;    -   (d) amplifying the adapter-ligated, preferably tagged,        restriction fragment hybridised with the primers by PCR or a        similar technique so as to cause further elongation of the        hybridised primers along the restriction fragments of the        starting DNA to which the primers hybridised; and    -   (e) detecting, identifying or recovering the amplified or        elongated DNA fragment thus obtained.

AFLP thus provides a reproducible subset of adaptor-ligated fragments.Other suitable methods for complexity reduction are Chromatine ImmunoPrecipitation (ChiP). This means that nuclear DNA is isolated, whilstproteins such as transcription factors are linked to the DNA. With ChiPfirst an antibody is used against the protein, resulting inAb-protein-DNA complex. By purifying this complex and precipitating it,DNA to which this protein binds is selected. Subsequently, the DNA canbe used for library construction and sequencing. I.e., this is a methodto perform a complexity reduction in a non-random fashion directed tospecific functional areas; in the present example specific transcriptionfactors.

One useful variant of the AFLP technology uses no selective nucleotides(i.c. +0/+0 primers) and is sometimes called linker PCR. This alsoprovides for a very suitable complexity reduction.

For a further description of AFLP, its advantages, its embodiments, aswell as the techniques, enzymes, adaptors, primers and further compoundsand tools used therein, reference is made to U.S. Pat. No. 6,045,994,EP-B-0 534 858, EP 976835 and EP 974672, WO01/88189 and Vos et al.Nucleic Acids Research, 1995, 23, 4407-4414, which are herebyincorporated in their entirety.

Thus, in a preferred embodiment of the method of the present invention,the complexity reduction is performed by

-   -   digesting the nucleic acid sample with at least one restriction        endonuclease to fragment it into restriction fragments;    -   ligating the restriction fragments obtained with at least one        double-stranded synthetic oligonucleotide adaptor having one end        compatible with one or both ends of the restriction fragments to        produce adapter-ligated restriction fragments;    -   contacting said adapter-ligated restriction fragments with one        or more oligonucleotide primers under hybridizing conditions;        and    -   amplifying said adapter-ligated restriction fragments by        elongation of the one or more oligonucleotide primers,

wherein at least one of the one or more oligonucleotide primers includea nucleotide sequence having the same nucleotide sequence as theterminal parts of the strands at the ends of said adapter-ligatedrestriction fragments, including the nucleotides involved in theformation of the target sequence for said restriction endonuclease andincluding at least part of the nucleotides present in the adaptors,wherein, optionally, at least one of said primers includes at its 3′ enda selected sequence comprising at least one nucleotide locatedimmediately adjacent to the nucleotides involved in the formation of thetarget sequence for said restriction endonuclease.

AFLP is a highly reproducible method for complexity reduction and istherefore particularly suited for the method according to the presentinvention.

In a preferred embodiment of the method according to the presentinvention, the adaptor or the primer comprises a tag. This isparticularly the case for the actual identification of thepolymorphisms, when it is important to distinguish between sequencesderived from separate libraries. Incorporating an oligonucleotide tag inan adaptor or primer is very convenient as no additional steps arerequired to tag a library.

In another embodiment, the tag is an identifier sequence. As discussedabove, such identifier sequence may be of varying length depending onthe amount of nucleic acid samples to be compared. A length of about 4bases (4⁴=256 different tag sequences possible) is sufficient todistinguish between the origin of a limited number of samples (up to256), although it is preferred that the tag sequences differ by morethan one base between the samples to be distinguished. As needed, thelength of the tag sequences can be adjusted accordingly.

In an embodiment, the sequencing is performed on a solid support such asa bead (see e.g. WO 03/004690, WO 03/054142, WO 2004/069849, WO2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of 454Corporation), which are herein incorporated by reference). Suchsequencing method is particularly suitable for cheap and efficientsequencing of many samples simultaneously.

In a preferred embodiment, the sequencing comprises the steps of:

-   -   annealing adapter-ligated fragments to beads, each bead being        annealed with a single adapter-ligated fragment;    -   emulsifying the beads in water-in-oil microreactors, each        water-in-oil microreactor comprising a single bead;    -   loading the beads in wells, each well comprising a single bead;        and    -   generating a pyrophosphate signal.

In the first step, sequencing adaptors are ligated to fragments withinthe combination library. Said sequencing adaptor includes at least a“key” region for annealing to a bead, a sequencing primer region and aPCR primer region. Thus, adapter-ligated fragments are obtained.

In a further step, adapter-ligated fragments are annealed to beads, eachbead annealing with a single adapter-ligated fragment. To the pool ofadapter-ligated fragments, beads are added in excess as to ensureannealing of one single adapter-ligated fragment per bead for themajority of the beads (Poisson distribution).

In a next step, the beads are emulsified in water-in-oil microreactors,each water-in-oil microreactor comprising a single bead. PCR reagentsare present in the water-in-oil microreactors allowing a PCR reaction totake place within the microreactors. Subsequently, the microreactors arebroken, and the beads comprising DNA (DNA positive beads) are enriched.

In a following step, the beads are loaded in wells, each well comprisinga single bead. The wells are preferably part of a PicoTiter™Plateallowing for simultaneous sequencing of a large amount of fragments.

After addition of enzyme-carrying beads, the sequence of the fragmentsis determined using pyrosequencing. In successive steps, thepicotiterplate and the beads as well as the enzyme beads therein aresubjected to different deoxyribonucleotides in the presence ofconventional sequencing reagents, and upon incorporation of adeoxyribonucleotide a light signal is generated which is recorded.Incorporation of the correct nucleotide will generate a pyrosequencingsignal which can be detected.

Pyrosequencing itself is known in the art and described inter alia onwww.biotagebio.com; www.pyrosequencing.com/tab technology The technologyis further applied in e.g. WO 03/004690, WO 03/054142, WO 2004/069849,WO 2004/070005, WO 2004/070007, and WO 2005/003375 (all in the name of454 Corporation), which are herein incorporated by reference.

The high-throughput screening of step k) is preferably performed byimmobilization of the probes designed in step h) onto an array, followedby contacting of the array comprising the probes with a test libraryunder hybridizing conditions. Preferably, the contacting step isperformed under stringent hybridizing conditions (see Kennedy et al.(2003) Nat. Biotech.; published online 7 Sep. 2003: 1-5). One skilled inthe art is aware of suitable methods for immobilization of probes ontoan array and of methods of contacting under hybridizing conditions.Typical technology that is suitable for this purpose is reviewed inKennedy et al. (2003) Nat. Biotech.; published online 7 Sep. 2003: 1-5.

One particular advantageous application is found in the breeding ofpolyploidal crops. By sequencing polyploidal crops with a high coverage,identifying SNPs and the various alleles and developing probes forallele-specific amplification significant progress can be made thebreeding of polyploidal crops.

As part of the invention, it has been found that the combination ofgenerating random selected subsets using selective amplification for aplurality of samples and high throughput sequencing technology presentscertain complex problems that had to be solved for the furtherimprovement of the herein described method for the efficient and highthroughput identification of polymorphisms. More in detail, it has beenfound that when multiple (i.e. the first and the second or further)samples are combined in a pool after performing a complexity reduction,a problem occurs that many fragments appear to be derived from twosamples, or, put differently, many fragments were identified that couldnot be allocated uniquely to one sample and thus could not be used inthe process of identifying polymorphisms. This lead to a reducedreliability of the method and to less polymorphisms (SNPs, indels, SSRs)that could be adequately identified.

After careful and detailed analysis of the entire nucleotide sequence ofthe fragments that could not be allocated, it was found that thosefragments contained two different tag-comprising adaptors and wereprobably formed between the generation of the complexity reduced samplesand the ligation of the sequencing adaptors. The phenomenon is depictedas ‘mixed tagging’. The phenomenon depicted as ‘mixed tagging’, as usedherein, thus refers to fragments that contain a tag relating thefragment to one sample on side, whereas the opposite side of thefragment contains a tag relating the fragment to another sample. Thusone fragment appears to be derived from two samples (quod non). Thisleads to erroneous identification of polymorphisms and is henceundesirable.

It has been theorised that the formation of heteroduplex fragmentsbetween two samples lies afoot to this anomaly.

The solution to this problem has been found in a redesigning of thestrategy for the conversion of samples of which the complexity isreduced to bead-annealed fragments that can be amplified prior to highthroughput sequencing. In this embodiment, each sample is subjected tocomplexity reduction and optional purification. After that, each sampleis rendered blunt (end-polishing) followed by ligation of the sequencingadapter that is capable of annealing to the bead. The sequencingadapter-ligated fragments of the samples are then combined and ligatedto the beads for emulsion polymerisation and subsequent high-throughputsequencing.

As a further part of this invention, it was found that the formation ofconcatamers hampered the proper identification of polymorphisms.Concatamers have been identified as fragments that are formed aftercomplexity reduction products have been ‘blunted’ or ‘polished’, forinstance by T4 DNA polymerase, and instead of ligating to the adaptersthat allow annealing to the beads, ligate to each other, hereby creatingconcatamers, i.e. a concatamer is the result of the dimerisation ofblunted fragments.

The solution to his problem was found in the use of certain specificallymodified adapters. The amplified fragments obtained from the complexityreduction typically contain a 3′-A overhang due to the characteristicsof certain preferred polymerases that do not have 3′-5′ exonucleaseproof-reading activity. The presence of such a 3′-A overhang is also thereason why fragments are blunted prior to adapter ligation. By providingan adapter that can anneal to a bead wherein the adapter contains a 3′-Toverhang, it was found that both the problem of the ‘mixed tags’ and ofthe concatamers can be solved in one step. A further advantage of usingthese modified adapters is that the conventional ‘blunting’ step and thesubsequent phosporylation step can be omitted.

Thus, in a further preferred embodiment, after the complexity reductionstep of each sample, a step is performed on the amplifiedadapter-ligated restriction fragments obtained from the complexityreduction step, whereby to these fragments sequencing adapters areligated, which sequencing adapters contain a 3′-T overhang and arecapable of annealing to the beads.

It has further been found that, when the primers used in the complexityreduction step are phosporylated, the end-polishing (blunting) step andintermediate phosporylation prior to ligation can be avoided.

Thus, in a highly preferred embodiment of the invention, the inventionrelates to a method for identifying one or more polymorphisms, saidmethod comprising the steps of:

-   -   a) providing a plurality of nucleic acid samples of interest;    -   b) performing a complexity reduction on each of the samples to        provide a plurality of libraries of the nucleic acid samples,        wherein the complexity reduction is performed by        -   digesting each nucleic acid sample with at least one            restriction endonuclease to fragment it into restriction            fragments;        -   ligating the restriction fragments obtained with at least            one double-stranded, synthetic oligonucleotide adaptor            having one end compatible with one or both ends of the            restriction fragments to produce adapter-ligated restriction            fragments;        -   contacting said adapter-ligated restriction fragments with            one or more phosporylated oligonucleotide primers under            hybridising conditions; and        -   amplifying said adapter-ligated restriction fragments by            elongation of the one or more oligonucleotide primers,            wherein at least one of the one or more oligonucleotide            primers include a nucleotide sequence having the same            nucleotide sequence as the terminal parts of the strands at            the ends of said adapter-ligated restriction fragments,            including the nucleotides involved in the formation of the            target sequence for said restriction endonuclease and            including at least part of the nucleotides present in the            adaptors, wherein, optionally, at least one of said primers            includes at its 3′ end a selected sequence comprising at            least one nucleotide located immediately adjacent to the            nucleotides involved in the formation of the target sequence            for said restriction endonuclease and wherein the adaptor            and/or the primer contain a tag;    -   c) combining said libraries to a combined library;    -   d) ligating sequencing adapters capable of annealing to beads to        the amplified adapter-capped fragments in the combined library,        using an sequencing adapter carrying a 3′-T overhang and        subjecting the bead-annealed fragments to emulsion        polymerisation;    -   e) sequencing of at least a portion of the combined library;    -   f) aligning the sequences from each sample obtained in step e);    -   g) determining one or more polymorphisms between the plurality        of nucleic acid samples in the alignment of step f);    -   h) using the one or more polymorphisms determined in step g) to        design detection probes;    -   i) providing a test sample nucleic acid of interest;    -   j) performing the complexity reduction of step b) on the test        sample nucleic acid of interest to provide a test library of the        test sample nucleic acid;    -   k) subjecting the test library to high-throughput screening to        identify the presence, absence or amount of the polymorphisms        determined in step g) using the detection probes designed in        step h).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the primer sequences for preamplification of PSP-11 andPI20234. FIG. 1A also shows a fragment according to the presentinvention annealed onto a bead (‘454 bead’) and the sequence of primerused for pre-amplification of the two pepper lines. ‘DNA fragment’denotes the fragment obtained after digestion with a restrictionendonuclease, ‘keygene adaptor’ denotes an adaptor providing anannealing site for the (phosphorylated) oligonucleotide primers used togenerate a library, ‘KRS’ denotes an identifier sequence (tag), ‘454SEQ. Adaptor’ denotes a sequencing adaptor, and ‘454 PCR adaptor’denotes an adaptor to allow for emulsion amplification of the DNAfragment. The PCR adaptor allows for annealing to the bead and foramplification and may contain a 3′-T overhang.

FIG. 1B shows a schematic primer used in the complexity reduction step.Such a primer generally comprises a recognition site region indicated as(2), a constant region that may include a tag section indicated as (1)and one or more selective nucleotides in a selective region indicated as(3) at the 3′-end thereof.).

FIGS. 2A and 2B show DNA concentration estimation using 2% agarosegel-electrophoresis. S1 denotes PSP11; S2 denote PI201234. 50, 100, 250and 500 ng denotes respectively 50 ng, 100 ng, 250 ng and 500 ng toestimate DNA amounts of S1 and S2. FIGS. 2C and 2D show DNAconcentration determination using Nanodrop spectrophotometry.

FIGS. 3A and 3B show the results of intermediate quality assessments ofexample 3. FIG. 3C shows DNA concentrations of each sample noted usingNanodrop.

FIG. 4A shows flow charts of the sequence data processing pipeline, i.e.the steps taken from the generation of the sequencing data to theidentification of putative SNPs, SSRs and indels, via steps of theremoval of known sequence information in Trimming & Tagging resulting intrimmed sequence data which are clustered and assembled to yield contigsand singletons (fragments that cannot be assembled in a contig) afterwhich putative polymorphisms can be identified and assessed. FIG. 4Bfurther elaborates on the process of polymorphisms mining.

FIGS. 5A, 5B and 5C address the problem of mixed tags and provides inpanel 1 an example of a mixed tag, carrying tags associated with sample1 (MS1) and sample 2 (MS2). Panel 2 provides a schematic explanation ofthe phenomenon. AFLP Restriction fragments derived from sample 1 (S1)and from sample 2 (S2) are ligated with adaptors (“Keygene adaptor”) onboth sides carrying sample specific tags S1 and S2. After amplificationand sequencing, e Expected fragments are those with S1-S1 tags and S2-S2tags. What additionally and unexpectedly is observed are also fragmentsthat carry tags S1-S2 or S2-S1. Panel 3 explains the hypothesized causeof the generation of mixed tags whereby heteroduplex products are formedfrom fragments from samples 1 and 2. The heteroduplexes aresubsequently, due to the 3′-5′ exonuclease activity of T4 DNA polymeraseor Klenow, rendered free from the 3′-protruding ends. Duringpolymerization, the gaps are filled with nucleotides and the incorrecttag is introduced. This works for heteroduplexes of about the samelength (top panel) but also for heteroduplexes of more varying length.Panel 4 provides on the right the conventional protocol leading to theformation of mixed tags and on the right the modified protocol.

FIGS. 6A, 6B and 6C address the problem of concatamer formation, wherebyin panel 1 a typical example of a concatamer is given, whereby thevarious adapter and tag sections are underlined and with their origin(i.e. MS1, MS2, ES1 and ES2 corresponding respectively to a MseIrestriction site-adapter from sample 1, MseI restriction site-adapterfrom sample 2, EcoRI restriction site-adapter from sample 1, EcoRIrestriction site-adapter from sample 2). Panel 2 demonstrates theexpected fragments carrying S1-S1 tags and S2-S2 tags and the observedbut unexpected S1-S1-S2-S2, being a concatamer of a fragments fromsample 1 and from sample 2. Panel 3 solution to avoid the generation ofconcatamers as well as mixed tags by introducing an overhang in the AFLPadaptors, modified sequencing adaptors and omission of the end-polishingstep when ligating sequencing adaptors. No concatamer formation is foundbecause the ALP fragments can not ligate to each other and no mixedfragments occur as the end-polishing step is omitted. Panel 4 providesthe modified protocol using modified adaptors to avoid concatamerformation as well as mixed tags.

FIG. 7. Multiple alignment “10037_CL989contig2” of pepper AFLP fragmentsequences, containing a putative single nucleotide polymorphism (SNP).Note that the SNP (indicated by an the black arrow) is defined by an Aallele present in both reads of sample 1 (PSP11), denoted by thepresence of the MS1 tag in the name of the top two reads, and a G allelepresent in sample 2 (PI201234), denoted by the presence of the MS2 tagin the name of the bottom two reads. Read names are shown on the left.The consensus sequence of this multiple alignment is (5′-3′):TAACACGACTTTGAACAAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACA[A/G]TGTTGGTTTTGGTGCTAACTTCAACCCCACTACTGTTTTGCTCTATTTTTG (SEQ ID NO: 47). FIG. 7discloses full-length sequences as SEQ ID NOS 64-68, respectively, inorder of appearance.

FIG. 8A. Schematic representation of enrichment strategy for targetingsimple sequence repeats (SSRs) in combination with high throughputsequencing for de novo SSR discovery.

FIG. 8B: Validation of a G/A SNP in pepper using SNPWave detection.P1=PSP11; P2=PI201234. Eight RIL offspring are indicated by numbers 1-8.

EXAMPLES Example 1

EcoRI/MseI restriction ligation mixture (1) was generated from genomicDNA of the pepper lines PSP-11 and PI20234. The restriction ligationmixture was 10 times diluted and 5 microliter of each sample waspre-amplified (2) with EcoRI +1(A) and MseI +1(C) primers (set I). Afteramplification the quality of the pre-amplification product of the twopepper samples was checked on a 1% agarose gel. The preamplificationproducts were 20 times diluted, followed by a KRSEcoRI +1(A) and KRSMseI+2(CA) AFLP pre-amplification. The KRS (identifier) sections areunderlined and the selective nucleotides are in bold at the 3′-end inthe primersequence SEQ ID 1-4 below. After amplification the quality ofthe pre-amplification product of the two pepper samples was checked on a1% agarose gel and by an EcoRI +3(A) and MseI +3(C) (3) AFLP fingerprint(4). The pre-amplification products of the two pepper lines wereseparately purified on a QiagenPCR column (5). The concentration of thesamples was measured on the nanodrop. A total of 5006.4 ng PSP-11 and5006.4 ng PI20234 was mixed and sequenced.

Primer set I used for preamplification of PSP-11

[SEQ ID 1] E01LKRS1 5′-CGTCAGACTGCGTACCAATTCA-3′ [SEQ ID 2] M15KKRS15′-TGGTGATGAGTCCTGAGTAACA-3′

Primer set II used for preamplification of PI20234

E01LKRS2 [SEQ ID 3] 5′-CAAGAGACTGCGTACCAATTCA-3′ M15KKRS2 [SEQ ID 4]5′-AGCCGATGAGTCCTGAGTAACA-3′

(1) EcoRI/MseI Restriction Ligation Mixture

Restriction Mix (40 ul/Sample)

DNA 6 μl (±300 ng) ECoRI (5 U) 0.1 μl MseI (2 U) 0.05 μl 5xRL 8 μl MQ25.85 μl Totaal 40 μl

Incubation during 1 h. at 37° C.

Addition of:

Ligation Mix (10 μl/Sample)

10 mM ATP 1 μl T4 DNA ligase 1 μl ECoRI adapt. (5 pmol/μl) 1 μl MseIadapt.. (50 pmol/μl) 1 μl 5xRL 2 μl MQ 4 μl Totaal 10 μl 

Incubation during 3 h. at 37° C.

EcoRI-Adaptor

[SEQ ID 5] 91M35/91M36: *-CTCGTAGACTGCGTACC     :91M35 [SEQ ID 6]± bio             CATCTGACGCATGGTTAA :91M36

MseI-Adaptor

[SEQ ID 7] 92A18/92A19: 5-GACGATGAGTCCTGAG-3   :92A18 [SEQ ID 8]                 3-TACTCAGGACTCAT-5 :92A19

(2) Pre-Amplification

Preamplification (A/C):

RL-mix (10x) 5 μl EcoRI-pr E01L (50 ng/ul) 0.6 μl MseI-pr M02K (50ng/ul) 0.6 μl dNTPs (25 mM) 0.16 μl Taq.pol. (5 U) 0.08 μl 10XPCR 2.0 μlMQ 11.56 μl Total 20 μl/reaction

Pre-Amplification Thermal Profile

Selective pre amplification was done in a reaction volume of 50 μl. ThePCR was performed in a PE GeneAmp PCR System 9700 and a 20 cycle profilewas started with a 94° C. denaturation step for 30 seconds, followed byan annealing step of 56° C. for 60 seconds and an extension step of 72°C. for 60 seconds.

EcoRI +1(A)¹

[SEQ ID 9] E01 L 92R11: 5-AGACTGCGTACCAATTCA-3

MseI +1(C)¹

[SEQ ID 10] M02k 93E42: 5-GATGAGTCCTGAGTAAC-3

Preamplification A/CA:

PA+1/+1-mix (20x) 5 μl EcoRI-pr 1.5 μl MseI-pr. 1.5 μl dNTPs (25 mM) 0.4μl Taq.pol. (5 U) 0.2 μl 10XPCR 5 μl MQ 36.3 μl Total 50 μl

Selective pre amplification was done in a reaction volume of 50 μl. ThePCR was performed in a PE GeneAmp PCR System 9700 and a 30 cycle profilewas started with a 94° C. denaturation step for 30 seconds, followed byan annealing step of 56° C. for 60 seconds and an extension step of 72°C. for 60 seconds.

(3) KRSEcoRI +1(A) and KRSMseI +2(CA)²

[SEQ ID 11] 05F212 E01LKRS1 CGTCAGACTGCGTACCAATTCA-3′ [SEQ ID 12] 05F213E01LKRS2 CAAGAGACTGCGTACCAATTCA-3′ [SEQ ID 13] 05F214 M15KKRS1TGGTGATGAGTCCTGAGTAACA-3′ [SEQ ID 14] 05F215 M15KKRS2AGCCGATGAGTCCTGAGTAACA-3′

selective nucleotides in bold and tags (KRS) underlined

Sample PSP11: E01LKRS1/M15KKRS1

Sample PI120234: E01LKRS2/M15KKRS2

(4) AFLP Protocol

Selective amplification was done in a reaction volume of 20 μl. The PCRwas performed in a PE GeneAmp PCR System 9700. A 13 cycle profile wasstarted with a 94° C. denaturation step for 30 seconds, followed by anannealing step of 65° C. for 30 seconds, with a touchdown phase in whichthe annealing temperature was lowered 0.7° C. in each cycle, and anextension step of 72° C. for 60 seconds. This profile was followed by a23 cycle profile with a 94° C. denaturation step for 30 seconds,followed by an annealing step of 56° C. for 30 seconds and an extensionstep of 72° C. for 60 seconds.

EcoRI +3(AAC) and MseI +3(CAG)

[SEQ ID 15] E32 92S02: 5-GACTGCGTACCAATTC AAC -3 [SEQ ID 16] M49 92G23:5-GATGAGTCCTGAGTAA CAG -3

(5) Qiagen Column

Qiagen purification was performed according to the manufacturer'sinstruction: QIAquick® Spin Handbook.

Example 2: PEPPER

DNA from the Pepper lines PSP-11 and PI20234 was used to generate AFLPproduct by use of AFLP Keygene Recognition Site specific primers. (TheseAFLP primers are essentially the same as conventional AFLP primers, e.g.described in EP 0 534 858, and will generally contain a recognition siteregion, a constant region and one or more selective nucleotides in aselective region.

From the pepper lines PSP-11 or PI20234 150 ng of DNA was digested withthe restriction endonucleases EcoRI (5 U/reaction) and MseI (2U/reaction) for 1 hour at 37° C. following by inactivation for 10minutes at 80° C. The obtained restriction fragments were ligated withdouble-stranded synthetic oligonucleotide adapter, one end of which iscompatible with one or both of the ends of the EcoRI and/or MseIrestriction fragments. AFLP preamplification reactions (20 μl/reaction)with +1/+1 AFLP primers were performed on 10 times dilutedrestriction-ligation mixture. PCR profile:20*(30 s at 94° C.+60 s at 56°C.+120 s at 72° C.). Additional AFLP reactions (50 μl/reaction) withdifferent +1 EcoRI and +2 MseI AFLP Keygene Recognition Site specificprimers (Table below, tags are in bold, selective nucleotides areunderlined.) were performed on 20 times diluted +1/+1 EcoRI/MseI AFLPpreamplification product. PCR profile: 30*(30 s at 94° C.+60 s at 56°C.+120 s at 72° C.). The AFLP product was purified by using the QIAquickPCR Purification Kit (QIAGEN) following the QIAquick® Spin Handbook07/2002 page 18 and the concentration was measured with a NanoDrop®ND-1000 Spectrophotometer. A total of 5 μg of +1/+2 PSP-11 AFLP productand 5 μg of +1/+2 PI20234 AFLP product was put together and solved in23.3 μl TE. Finally a mixture with a concentration of 430 ng/μl +1/+2AFLP product was obtained.

TABLE AFLP SEQ ID PCR primer Primer-3′ Pepper reaction [SEQ ID 17] 05F21CGTCAGACTGCGTACCAATTCA PSP- 1 [SEQ ID 18] 05F21 TGGTGATGAGTCCTGAGTAACAPSP- 1 [SEQ ID 19] 05F21 CAAGAGACTGCGTACCAATTCA PI2023 2 [SEQ ID 20]05F21 AGCCGATGAGTCCTGAGTAACA PI2023 2

Example 3: Maize

DNA from the Maize lines B73 and M017 was used to generate AFLP productby use of AFLP Keygene Recognition Site specific primers. (These AFLPprimers are essentially the same as conventional AFLP primers, e.g.described in EP 0 534 858, and will generally contain a recognition siteregion, a constant region and one or more selective nucleotides at the3′-end thereof.).

DNA from the pepper lines B73 or M017 was digested with the restrictionendonucleases TaqI (5 U/reaction) for 1 hour at 65° C. and MseI (2U/reaction) for 1 hour at 37° C. following by inactivation for 10minutes at 80° C. The obtained restriction fragments were ligated withdouble-stranded synthetic oligonucleotide adapter, one end of which iscompatible with one or both of the ends of the TaqI and/or MseIrestriction fragments.

AFLP preamplification reactions (20 μl/reaction) with +1/+1 AFLP primerswere performed on 10 times diluted restriction-ligation mixture. PCRprofile:20*(30 s at 94° C.+60 s at 56° C.+120 s at 72° C.). AdditionalAFLP reactions (50 μl/reaction) with different +2 TaqI and MseI AFLPKeygene Recognition Site primers (Table below, tags are in bold,selective nucleotides are underlined.) were performed on 20 timesdiluted +1/+1 TaqI/MseI AFLP preamplification product. PCR profile:30*(30 s at 94° C.+60 s at 56° C.+120 s at 72° C.). The AFLP product waspurified by using the QIAquick PCR Purification Kit (QIAGEN) followingthe QIAquick® Spin Handbook 07/2002 page 18 and the concentration wasmeasured with a NanoDrop® ND-1000 Spectrophotometer. A total of 1.25 μgof each different B73 +2/+2 AFLP product and 1.25 μg of each differentM017 +2/+2 AFLP product was put together and solved in 30 μl TE. Finallya mixture with a concentration of 333 ng/μl +2/+2 AFLP product wasobtained.

TABLE AFLP SEQ ID PCR Primer Primer sequence Maize Reaction [SEQ ID 21]05G360 ACGTGTAGACTGCGTACCGAAA B73 1 [SEQ ID 22] 05G368ACGTGATGAGTCCTGAGTAACA B73 1 [SEQ ID 23] 05G362 CGTAGTAGACTGCGTACCGAACB73 2 [SEQ ID 24] 05G370 CGTAGATGAGTCCTGAGTAACA B73 2 [SEQ ID 25] 05G364GTACGTAGACTGCGTACCGAAG B73 3 [SEQ ID 26] 05G372 GTACGATGAGTCCTGAGTAACAB73 3 [SEQ ID 27] 05G366 TACGGTAGACTGCGTACCGAAT B73 4 [SEQ ID 28] 05G374TACGGATGAGTCCTGAGTAACA B73 4 [SEQ ID 29] 05G361 AGTCGTAGACTGCGTACCGAAAM017 5 [SEQ ID 30] 05G369 AGTCGATGAGTCCTGAGTAACA M017 5 [SEQ ID 31]05G363 CATGGTAGACTGCGTACCGAAC M017 6 [SEQ ID 32] 05G371CATGGATGAGTCCTGAGTAACA M017 6 [SEQ ID 33] 05G365 GAGCGTAGACTGCGTACCGAAGM017 7 [SEQ ID 34] 05G373 GAGCGATGAGTCCTGAGTAACA M017 7 [SEQ ID 35]05G367 TGATGTAGACTGCGTACCGAAT M017 8 [SEQ ID 36] 05G375TGATGATGAGTCCTGAGTAACA M017 8

Finally the 4 P1-samples and the 4 P2-samples were pooled andconcentrated. A total amount of 25 μl of DNA product and a finalconcentration of 400 ng/ul (total of 10 μg) was obtained. Intermediatequality assessments are given in FIG. 3.

Sequencing by 454

Pepper and maize AFLP fragment samples as prepared as describedhereinbefore were processed by 454 Life Sciences as described (Margulieset al., 2005. Genome sequencing in microfabricated high-densitypicoliter reactors. Nature 437 (7057):376-80. Epub Jul. 31, 2005).

Data Processing

Processing Pipeline:

Input Data

raw sequence data were received for each run:

-   -   200,000-400,000 reads    -   base calling quality scores        Trimming and Tagging

These sequence data are analyzed for the presence of Keygene RecognitionSites (KRS) at the beginning and end of the read. These KRS sequencesconsist of both AFLP-adaptor and sample label sequence and are specificfor a certain AFLP primer combination on a certain sample. The KRSsequences are identified by BLAST and trimmed and the restriction sitesare restored. Reads are marked with a tag for identification of the KRSorigin. Trimmed sequences are selected on length (minimum of 33 nt) toparticipate in further processing.

Clustering and Assembly

A MegaBlast analysis is performed on all size-selected, trimmed reads toobtain clusters of homologous sequences. Consecutively all clusters areassembled with CAPS to result in assembled contigs. From both stepsunique sequence reads are identified that do not match any other reads.These reads are marked as singletons.

The processing pipeline carrying out the steps described herein beforeis shown in FIG. 4A

Polymorphism Mining and Quality Assessment

The resulting contigs from the assembly analysis form the basis ofpolymorphism detection. Each ‘mismatch’ in the alignment of each clusteris a potential polymorphism. Selection criteria are defined to obtain aquality score:

-   -   number of reads per contig    -   frequency of ‘alleles’ per sample    -   occurrence of homopolymer sequence    -   occurrence of neighbouring polymorphisms

SNPs and indels with a quality score above the threshold are identifiedas putative polymorphisms. For SSR mining we used the MISA(MlcroSAtellite identification) tool. This tool identifies di-, tri-,tetranucleotide and compound SSR motifs with predefined criteria andsummarizes occurrences of these SSRs.

The polymorphism mining and quality assignment process is shown in FIG.4B

Results

The table below summarizes the results of the combined analysis ofsequences obtained from 2 454 sequence runs for the combined peppersamples and 2 runs for the combined maize samples.

Pepper Maize Total number of reads 457178 492145 Number of trimmed reads399623 411008 Number singletons 105253 313280 Number of contigs 3186314588 Number of reads in contigs 294370 97728 Total number of sequencescontaining SSRs 611 202 Number of different SSR-containing sequences 10465 Number of different SSR motifs (di, tri, tetra and 49 40 compound)Number SNPs with Q score ≧0.3* 1636 782 Number of indels* 4090 943 *bothwith selection against neighboring SNPs, at least 12 bp flankingsequence and not occurring in homopolymer sequences larger than 3nucleotides.

Example 4. Single Nucleotide Polymorphism (SNP) Discovery in Pepper

DNA Isolation

Genomic DNA was isolated from the two parental lines of a pepperrecombinant inbred (RIL) population and 10 RIL progeny. The parentallines are PSP11 and PI201234. Genomic DNA was isolated from leafmaterial of individual seedlings using a modified CTAB proceduredescribed by Stuart and Via (Stuart, C. N., Jr and Via, L. E. (1993) Arapid CTAB DNA isolation technique useful for RAPD fingeprinting andother PCR applications. Biotechniques, 14, 748-750). DNA samples werediluted to a concentration of 100 ng/μl in TE (10 mM Tris-HCl pH 8.0, 1mM EDTA) and stored at −20° C.

AFLP Template Preparation Using Tagged AFLP Primers

AFLP templates of the pepper parental lines PSP11 and PI201234 wereprepared using the restriction endonuclease combination EcoRI/MseI asdescribed by Zabeau & Vos, 1993: Selective restriction fragmentamplification; a general method for DNA fingerprinting. EP 0534858-A1,B1; U.S. Pat. No. 6,045,994) and Vos et al (Vos, P., Hogers, R.,Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot,J., Peleman, J., Kuiper, M. et al. (1995) AFLP: a new technique for DNAfingerprinting. Nucl. Acids Res., 21, 4407-4414).

Specifically, restriction of genomic DNA with EcoRI and MseI was carriedout as follows:

DNA Restriction

DNA 100-500 ng EcoRI 5 units MseI 2 units 5xRL buffer 8 μl MilliQ waterto 40 μl

Incubation was for 1 hour at 37° C. After the enzyme restriction,enzymes were inactivated by incubation for 10 minutes at 80° C.

Ligation of Adapters

10 mM ATP 1 μl T4 DNA ligase 1 μl EcoRI adaptor (5 pmol/μl) 1 μl MseIadaptor (50 pmol/μl) 1 μl 5xRL buffer. 2 μl MilliQ water to 40 μl

Incubation was for 3 hours at 37° C.

Selective AFLP Amplification

Following restriction-ligation, the restriction/ligation reaction wasdiluted 10-fold with T₁₀E_(0.1) and 5 μl diluted mix was used as atemplate in a selective amplification step. Note that since a +1/+2selective amplification was intended, first a +1/+1 selectivepre-amplification step (with standard AFLP primers) was performed.Reaction conditions of the +1/+1 (+A/+C) amplification were as follows.

Restriction-Ligation mix (10-fold diluted) 5 μl EcoRI-primer +1 (50ng/μl): 0.6 μl MseI-primer +1 (50 ng/μl) 0.6 μl dNTPs (20 mM) 0.2 μlTaq.polymerase (5 U/μl Amplitaq, PE) 0.08 μl 10XPCR buffer 2.0 μl MilliQwater to 20 μl

Primers sequences were:

EcoRI + 1: [SEQ ID 9] 5′-AGACTGCGTACCAATTCA-3′ and MseI + 1: [SEQ ID 10]5′-GATGAGTCCTGAGTAAC-3′

PCR amplifications were performed using a PE9700 with a gold or silverblock using the following conditions: 20 times (30 s at 94° C., 60 s at56° C. and 120 s at 72° C.)

The quality of the generated +1/+1 preamplification products was checkedon a 1% agarose gel using a 100 basepair ladder and a 1 Kb ladder tocheck the fragment length distribution. Following +1/+1 selectiveamplification, the reaction was diluted 20-fold with T₁₀E_(0.1) and 5 μldiluted mix is used as a template in the +1/+2 selective amplificationstep using tagged AFLP primers.

Finally, +1/+2 (A/+CA) selective AFLP amplifications were performed:

+1/+1 selective amplification product (20-fold diluted) 5.0 μl KRSEcoRI-primer +A (50 ng/μl) 1.5 μl KRS MseI-primer +CA (50 ng/μl) 1.5 μldNTPs (20 mM) 0.5 μl Taq polymerase (5 U/μl Amplitaq, Perkin Elmer) 0.2μl 10X PCR buffer 5.0 μl MQ to 50 μlTagged AFLP primers sequences were:

PSP11: [SEQ ID 1] O5F212: EcoRI + 1: 5′-CGTCAGACTGCGTACCAATTCA-3′ and[SEQ ID 2] O5F214: MseI + 2: 5′-TGGTGATGAGTCCTGAGTAACA-3′ PI201234: [SEQID 3] 05F213: EcoRI + 1: 5′-CAAGAGACTGCGTACCAATTCA-3′ and [SEQ ID 4]05F215: MseI + 1: 5′-AGCCGATGAGTCCTGAGTAACA-3′

Note that these primers contain 4 bp tags (underlined above) at their 5prime ends to distinguish amplification products originating from therespective pepper lines at the end of the sequencing process.

Schematic representation of pepper AFLP +1/+2 amplification productsafter amplification with AFLP primers containing 4 bp 5 prime tagsequences.

EcoRI tag MseI tag PSP 11: 5′-CGTC -------------------------------------ACCA-3′ 3′-GCAG--------------------------------------- TGGT-5′ PI2012345′-CAAG --------------------------------- GGCT-3′ 3′-GTTC--------------------------------- CCGA-5′

PCR amplifications (24 per sample) were performed using a PE9700 5 witha gold or silver block using the following conditions: 30 times (30 s at94° C.+60 s at 56° C.+120 s at 72° C.)

The quality of the generated amplification products was checked on a 1%agarose gel using a 100 basepair ladder and a 1 Kb ladder to check thefragment length distribution.

AFLP Reaction Purification and Quantification.

After pooling two 50 microliter +1/+2 selective AFLP reactions perpepper sample, the resulting 12 100 μl AFLP reaction products werepurified using the QIAquick PCR Purification Kit (QIAGEN), following theQIAquick® Spin handbook (Page 18). On each column a maximum of 100 μlproduct was loaded. Amplified products were eluted in T₁₀E_(0.1). Thequality of the purified products is checked on a 1% agarose gel andconcentrations were measured on the Nanodrop (FIG. 2).

Nanodrop concentration measurements were used to adjust the finalconcentration of each purified PCR product to 300 nanograms permicroliter. Five micrograms purified amplified product of PSP11 and 5microgram of PI201234 were mixed to generate 10 microgram templatematerial for preparation of the 454 sequencing library.

Sequence Library Preparation and High-Throughput Sequencing

Mixed amplification products from both pepper lines were subjected tohigh-throughput sequencing using 454 Life Sciences sequencing technologyas described by Margulies et al., (Margulies et al., Nature 437, pp.376-380 and Online Supplements). Specifically, the AFLP PCR productswere first end-polished and subsequently ligated to adaptors tofacilitate emulsion-PCR amplification and subsequent fragment sequencingas described by Margulies and co-workers. 454 adaptor sequences,emulsion PCR primers, sequence-primers and sequence run conditions wereall as described by Margulies and co-workers. The linear order offunctional elements in an emulsion-PCR fragment amplified on Sepharosebeads in the 454 sequencing process was as follows as exemplified inFIG. 1A:

454 PCR adaptor—454 sequence adaptor—4 bp AFLP primer tag 1—AFLP primersequence 1 including selective nucleotide(s)—AFLP fragment internalsequence—AFLP primer sequence 2 including selective nucleotide(s), 4 bpAFLP primers tag 2-454 sequence adaptor—454 PCR adaptor—Sepharose bead

Two high-throughput 454 sequence runs were performed by 454 LifeSciences (Branford, Conn.; United States of America).

454 Sequence Run Data-Processing.

Sequence data resulting from 2 454 sequence runs were processed using abio-informatics pipeline (Keygene N.V.). Specifically, raw 454basecalled sequence reads were converted in FASTA format and inspectedfor the presence of tagged AFLP adaptor sequences using a BLASTalgorithm. Upon high-confidence matches to the known tagged AFLP primersequences, sequences were trimmed, restriction endonuclease sitesrestored and assigned the appropriate tags (sample 1 EcoRI (ES1), sample1 MseI (MS1), sample 2 EcoRI (ES2) or sample 2 MseI (MS2),respectively). Next, all trimmed sequences larger than 33 bases wereclustered using a megaBLAST procedure based on overall sequencehomologies. Next, clusters were assembled into one or more contigsand/or singletons per cluster, using a CAP3 multiple alignmentalgorithm. Contigs containing more than one sequence were inspected forthe sequence mismatches, representing putative polymorphisms. Sequencemismatches were assigned quality scores based on the following criteria:

-   -   the numbers of reads in a contig    -   the observed allele distribution        -   The above two criteria form the basis for the so called Q            score assigned to each putative SNP/indel. Q scores range            from 0 to 1; a Q score of 0.3 can only be reached in case            both alleles are observed at least twice.    -   location in homopolymers of a certain length (adjustable;        default setting to avoid polymorphism located in homopolymers of        3 bases or longer).    -   number of contigs in cluster.    -   distance to nearest neighboring sequence mismatches (adjustable;        important for certain types of genotyping assays probing        flanking sequences)    -   the level of association of observed alleles with sample 1 or        sample 2; in case of a consistent, perfect association between        the alleles of a putative polymorphism and samples 1 and 2, the        polymorphism (SNP) is indicated as an “elite” putative        polymorphism (SNP). An elite polymorphism is thought to have a        high probability of being located in a unique or low-copy genome        sequence in case two homozygous lines have been used in the        discovery process. Conversely, a weak association of a        polymorphism with sample origin bears a high risk of having        discovered false polymorphisms arising from alignment of        non-allelic sequences in a contig.

Sequences containing SSR motifs were identified using the MISA searchtool.

Overall statistics of the run is shown in the Table below.

TABLE Overall statistics of a 454 sequence run for SNP discovery inpepper. Enzyme combination Run Trimming All reads 254308 Fault  5293(2%) Correct 249015 (98%) Concatamers   2156 (8.5%) Mixed tags   1120(0.4%) Correct reads Trimmed one end 240817 (97%) Trimmed both ends 8198 (3%) Number of reads sample 1 136990 (55%) Number of reads sample2 112025 (45%) Clustering Number of contigs 21918 Reads in contigs190861 Average number reads per contig 8.7 SNP mining SNPs with Q score≧0.3* 1483 Indel with Q score ≧0.3* 3300 SSR mining Total number of SSRmotifs identified 359 Number of reads containing one or more SSR 353motifs Number of SSR motif with unit size 1 0 (homopolymer) Number ofSSR motif with unit size 2 102 Number of SSR motif with unit size 3 240Number of SSR motif with unit size 4 17 *SNP/indel mining criteria wereas follows: No neighbouring polymorphisms with Q score larger than 0.1within 12 bases on either side, not present in homopolymers of 3 or morebases. Mining criteria did not take into account consistent associationwith sample 1 and 2, i.e. the SNPs and indels are not necessarily eliteputative SNPs/indels

An example of a multiple alignment containing an elite putative singlenucleotide polymorphism is shown in FIG. 7.

Example 5. SNP Validation by PCR Amplification and Sanger Sequencing

In order to validate the putative A/G SNP identified in example 1, asequence tagged site (STS) assay for this SNP was designed usingflanking PCR primers. PCR primer sequences were as follows:

Primer_1.2f: [SEQ ID 37] 5′-AAACCCAAACTCCCCCAATC-3′, and Primer_1.2r:[SEQ ID 38] 5′-AGCGGATAACAATTTCACACAGGA CATCAGTAGTCACACTGGTACAAAAATAGAGCAAAACAGTAGTG-3′

Note that primer 1.2r contained an M13 sequence primer binding site andlength stuffer at its 5 prime end. PCR amplification was carried outusing +A/+CA AFLP amplification products of PSP11 and PI210234 preparedas described in example 4 as template. PCR conditions were as follows:

For 1 PCR reaction the following components were mixed:

5 μl 1/10 diluted AFLP mixture (app. 10 ng/μl) 5 μl 1 pmol/μl primer1.2f (diluted directly from a 500 μM stock) 5 μl 1 pmol/μl primer 1.2r(diluted directly from a 500 μM stock) 5 μl PCR mix 2 μl 10 x PCR buffer1 μl 5 mM dNTPs 1.5 μl 25 mM MgCl₂ 0.5 μl H₂O 5 μl Enzyme mix 0.5 μl 10x PCR buffer (Applied Biosystems) 0.1 μl 5 U/μl AmpliTaq DNA polymerase(Applied Biosystems) 4.4 μl H₂OThe following PCR profile was used:

Cycle 1  2′; 94° C. Cycle 2-34 20″; 94° C. 30″; 56° C. 2′30″; 72° C.Cycle 35  7′; 72° C. ∞;  4° C.

PCR products were cloned into vector pCR2.1 (TA Cloning kit; Invitrogen)using the TA Cloning method and transformed into INVαT competent E. colicells. Transformants were subjected to blue/white screening. Threeindependent white transformants each for PSP11 and PI-201234 wereselected and grown 0/N in liquid selective medium for plasmid isolation.

Plasmids were isolated using the QIAprep Spin Miniprep kit (QIAGEN).Subsequently, the inserts of these plasmids were sequenced according tothe protocol below and resolved on the MegaBACE 1000 (Amersham).Obtained sequences were inspected on the presence of the SNP allele. Twoindependent plasmids containing the PI-201234 insert and 1 plasmidcontaining the PSP11 insert contained the expected consensus sequenceflanking the SNP. Sequence derived from the PSP11 fragment contained theexpected A (underlined) allele and sequence derived from PI-201234fragment contained the expected G allele (double underlined):

PSP11 (sequence 1): (5′-3′) [SEQ ID 39]AAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACAATGTTGGTTTTGGTGCTAACTTCAACCCCACTACTGTTTTGCTCTATTTTTGT PI-201234 (sequence 1):(5′-3′) [SEQ ID 40] AAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACAGTGTTGGTTTTGGTGCTAACTTCAACCCCACTACTGTTTTGCTCTATTTTTG PI-201234 (sequence 2): (5′-3′)[SEQ ID 41] AAACCCAAACTCCCCCAATCGATTTCAAACCTAGAACA G TGTTGGTTTTGGTGCTAACTTCAACCCCACTACTGTTTTGCTCTATTTTTG

This result indicates that the putative pepper A/G SNP represents a truegenetic polymorphism detectable using the designed STS assay.

Example 6: SNP Validation by SNPWave Detection

In order to validate the putative A/G SNP identified in example 1,SNPWave ligation probes sets were defined for both alleles of this SNPusing the consensus sequence. Sequence of the ligation probes were asfollows:

SNPWave probe sequences (5′-3′): [SEQ ID 42] 06A162GATGAGTCCTGAGTAACCCAATCGATTTCAAACCTAGAACAA (42 bases) [SEQ ID 43] 06A163GATGAGTCCTGAGTAACCACCAATCGATTTCAAACCTAGAACA G (44 bases) [SEQ ID 44]06A164 Phosphate-TGTTGGTTTTGGTGCTAACTTCAACCAACATCT GGAATTGGTACGCAGTC (52bases)

Note the allele specific probes 06A162 and 06A163 for the A and Galleles, respectively, differ by 2 bases in size, such that uponligation to the common locus-specific probe 06A164, ligation productsizes of 94 (42+54) and 96 (44+52) bases result.

SNPWave ligation and PCR reactions were carried as described by Van Eijkand co-workers (M. J. T. van Eijk, J. L. N. Broekhof, H. J. A. van derPoel, R. C. J. Hogers, H. Schneiders, J. Kamerbeek, E. Verstege, J. W.van Aart, H. Geerlings, J. B. Buntjer, A. J. van Oeveren, and P. Vos.(2004). SNPWave™: a flexible multiplexed SNP genotyping technology.Nucleic Acids Research 32: e47), using 100 ng genomic DNA of pepperlines PSP11 and PI201234 and 8 RIL offspring as starting material.Sequences of the PCR primers were:

93L01FAM (E00k): 5-GACTGCGTACCAATTC-3′[SEQ ID 45]

93E40 (MOOk): 5-GATGAGTCCTGAGTAA-3′[SEQ ID 46]

Following PCR amplification, PCR product purification and detection onthe MegaBACE1000 was as described by van Eijk and co-workers (videsupra). A pseudo-gel image of the amplification products obtained fromPSP11, PI201234 and 8 RIL offspring is shown in FIG. 8B.

The SNPWave results demonstrate clearly that the A/G SNP is detected bythe SNPWave assay, resulting in 92 bp products (=AA homozygous genotype)for P1 (PSP11) and RIL offspring 1, 2, 3, 4, 6 and 7), and in 94 bpproducts (=GG homozygous genotype) for P2 (PI201233) and RIL offspring 5and 8.

Example 7: Strategies for Enriching AFLP Fragment Libraries for Low-CopySequences

This example describes several enrichment methods to target low-copy ofunique genome sequences in order to increase the yield of elitepolymorphisms such as described in example 4. The methods can be dividedinto four categories:

1) Methods Aimed at Preparing High-Quality Genomic DNA, ExcludingChloroplast Sequences.

Here it is proposed to prepare nuclear DNA instead of whole genomic DNAas described in Example 4, to exclude co-isolation of abundantchloroplast DNA, which may result in reduced number of plant genomic DNAsequences, depending on the restriction endonucleases and selective AFLPprimers used in the fragment library preparation process. A protocol forisolation of highly pure tomato nuclear DNA has been described byPeterson, DG., Boehm, K. S. & Stack S. M. (1997). Isolation of MilligramQuantities of Nuclear DNA From Tomato (Lycopersicon esculentum), A PlantContaining High Levels of Polyphenolic Compounds. Plant MolecularBiology Reporter 15 (2), pages 148-153.

2) Methods Aimed at Using Restriction Endonucleases in the AFLP TemplatePreparation Process which are Expected to Yield Elevated Levels ofLow-Copy Sequences.

Here it is proposed to use certain restriction endonucleases in the AFLPtemplate preparation process, which are expected to target low-copy orunique genome sequences, resulting in fragment libraries enriched forpolymorphisms with increased ability to be convertible into genotypingassays. An examples of a restriction endonuclease targeting low-copysequence in plant genomes is PstI. Other methylation sensitiverestriction endonucleases may also target low-copy or unique genomesequences preferentially.

3) Methods Aimed a Selectively Removing Highly Duplicated SequencesBased on Re-Annealing Kinetics of Repeat Sequences Versus Low-CopySequences.

Here it is proposed to selectively remove highly duplicated (repeat)sequences from either the total genomic DNA sample or from the(cDNA-)AFLP template material prior to selective amplification.

3a) High-C₀t DNA preparation is a commonly used technique to enrichslowly annealing low-copy sequences from a complex plant genomic DNAmixture (Yuan et al. 2003; High-Cot sequence analysis of the maizegenome. Plant J. 34: 249-255). It is suggested to take High-C₀t insteadof total genomic DNA as starting material to enrich for polymorphismslocated in low-copy sequences.

3b) An alternative to laborious high-C₀t preparation may be incubatedenatured and re-annealing dsDNA with a novel nuclease from theKamchatka crab, which cleaves short, perfectly matched DNA duplexes at ahigher rate than nonperfectly matched DNA duplexes, as described byZhulidov and co-workers (2004; Simple cDNA normalization using Kamchatkacrab duplex-specific nuclease. Nucleic Acids Research 32, e37) andShagin and co-workers (2006; a novel method for SNP detection using anew duplex-specific nuclease from crab hepatopancreas. Genome Research12: 1935-1942). Specifically, it is proposed to incubate AFLPrestriction/ligation mixtures with this endonuclease to deplete themixture of highly duplicated sequences, followed by selective AFLPamplification of the remaining low-copy or unique genome sequences.

3c) Methyl filtration is a method to enrich for hypomethylated genomicDNA fragments using the restriction endonuclease McrBC which cutsmethylated DNA in the sequence [A/G]C, where the C is methylated (seePablo D. Rabinowicz, Robert Citek, Muhammad A. Budiman, Andrew Nunberg,Joseph A. Bedell, Nathan Lakey, Andrew L. O'Shaughnessy, Lidia U.Nascimento, W. Richard McCombie and Robert A. Martienssen. Differentialmethylation of genes and repeats in land plants. Genome Research15:1431-1440, 2005). McrBC may be used to enrich the low-copy sequencefraction of a genome as starting material for polymorphism discovery.

4) The Use of cDNA as Opposed to Genomic DNA in Order to Target GeneSequences.

Finally, here it is proposed to use oligodT-primed cDNA as opposed togenomic DNA as starting material for polymorphism discovery, optionallyin combination with the use the Crab duplex-specific nuclease describedin 3b above for normalization. Note that the use of oligodT primed cDNAalso excludes chloroplast sequences. Alternatively, cDNA-AFLP templatesinstead of oligodT primed cDNA is used to facilitate amplification ofthe remaining low-copy sequences in analogy to AFLP (see also 3b above).

Example 8: Strategy for Simple-Sequence Repeat Enrichment

This example describes the proposed strategy for discovery of SimpleSequence repeats sequences, in analogy to SNP discovery described inExample 4.

Specifically, Restriction-ligation of genomic DNA of two or more samplesis performed, e.g. using restriction endonucleases PstI/MseI. SelectiveAFLP amplification is performed as described in Example 4. Nextfragments containing the selected SSR motifs are enriched by one of twomethods:

1) Southern blot hybridization onto filters containing oligonucleotidesmatching the intended SSR motifs (e.g. (CA)₁₅ in case of enrichment forCA/GT repeats), followed by amplification of bound fragments in asimilar fashion as described by Armour and co-workers (Armour, J.,Sismani, C., Patsalis, P., and Cross, G. (2000). Measurement of locuscopy number by hybridization with amplifiable probes. Nucleic AcidsResearch vol 28, no. 2, pp. 605-609) or by

2) enrichment using biotinylated capture oligonucleotide hybridizationprobes to capture (AFLP) fragments in solution as described by Kijas andco-workers (Kijas, J. M., Fowler, J. C., Garbett C. A., and Thomas, M.R., (1994). Enrichment of microsatellites from the citrus genome usingbiotinylated oligonucleotide sequences bound to streptavidin-coatedmagnetic particles. Biotechniques, vol. 16, pp. 656-662.

Next, the SSR-motif enriched AFLP fragments are amplified using the sameAFLP primers are used in the preamplification step, to generate asequence library. An aliqout of the amplified fragments are T/A clonedand 96 clones are sequences to estimate the fraction of positive clones(clones containing the intended SSR motif, e.g. CA/GT motifs longer than5 repeat units. Another aliquot of the enriched AFLP fragment mixture isdetected by polyacrylamide gel electrophoresis (PAGE), optionally afterfurther selective amplification to obtain a readable fingerprint, inorder to visually inspect whether SSR containing fragments are enriched.Following successful completion of these control steps, the sequencelibraries are subjected to high-throughput 454 sequencing.

The above strategy for de novo SSR discovery is schematically depictedin FIG. 8A, and can be adapted for other sequence motifs by substitutingthe capture oligonucleotide sequences accordingly.

Example 9. Strategy for Avoiding Mixed Tags

Mixed tags refers to the observation that besides the expected taggedAFLP primer combination per sample, a low fraction of sequences areobserved which contain a sample 1 tag at one end, and a sample 2 tag athe other end (See also the table 1 in example 4). Schematically, theconfiguration of sequences containing mixed tags is depicted here-inbelow.

Schematic representation of the expected sample tag combinations.

EcoRI tag MseI tag PSP 11:5′-CGTC ------------------------------------------- ACCA-3′3′-GCAG-------------------------------------------- TGGT-5′ PI-2012345′-CAAG --------------------------------------- GGCT-3′ 3′-GTTC--------------------------------------- CCGA-5′Schematic representation of the mixed tags.

EcoRI tag MseI tag 5′-CGTC ------------------------------------------GGCT-3′ 3′-GCAG------------------------------------------- CCGA-5′5′-CAAG ------------------------------------------ ACCA-3′ 3′-GTTC----------------------------------------- TGGT-5′The observation of mixed tags precludes correct assignment of thesequence to either PSP11 or P1-201234.

An example of a mixed tag sequence observed in the pepper sequence rundescribed in Example 4 is shown in FIG. 5A. An overview of theconfiguration of observed fragments containing expected tags and mixedtags is shown in panel 2 of FIG. 5A.

The proposed molecular explanation for mixed tags is that during thesequence library preparation step, DNA fragments are made blunt by usingT4 DNA polymerase or Klenow enzyme to remove 3 prime protruding ends,prior to adaptor ligation (Margulies et al., 2005). While this may workwell when a single DNA sample is processed, in case of using a mixtureof two or more samples differently tagged DNA samples, fill in by thepolymerase results in incorporation of the wrong tag sequence in casewhen a heteroduplex has been formed between the complementary strandsderived from different samples (FIG. 5B panel 3 mixed tags) The solutionhas been found to pool samples after the purification step that followedadaptor ligation in the 454 sequence library construction step as shownin FIG. 5C panel 4.

Example 10. Strategy for Avoiding Mixed Tags and Concatamers Using anImproved Design for 454 Sequence Library Preparation

Besides the observation of low frequencies of sequence reads containingmixed tags as described in Example 9, a low frequency of sequence readsobserved from concatenated AFLP fragments have been observed.

An example of a sequence read derived from a concatamer is shown in FIG.6A Panel 1. Schematically, the configuration of sequences containingexpected tags and concatamers is shown in FIG. 6A Panel 2.

The proposed molecular explanation for the occurrence of concatenatedAFLP fragments is that during the 454 sequence library preparation step,DNA fragments are made blunt using T4 DNA polymerase or Klenow enzyme toremove 3 prime protruding ends, prior to adaptor ligation (Margulies etal., 2005). As a result, blunt end sample DNA fragments are incompetition with the adaptors during the ligation step and may beligated to each other prior to being ligated to adaptors. Thisphenomenon is in fact independent of whether a single DNA sample or amixture of multiple (tagged) samples are included in the librarypreparation step, and may therefore also occur during the conventionalsequencing as described by Margulies and co-workers. In case of theusing multiple tagged samples as described in Example 4, concatamerscomplicate correct assignment of sequence reads to samples based on thetag information and are therefore to be avoided.

The proposed solution to the formation of concatamers (and mixed tags)is to replace blunt-end adaptor ligation with ligation of adaptorscontaining a 3 prime T overhang, in analogy to T/A cloning of PCRproducts, as shown in FIG. 6B Panel 3. Conveniently, these modified3′prime T overhang-containing adaptors are proposed to contain a Coverhang at the opposite 3′end (which will not be ligated to the sampleDNA fragment, to prevent blunt-end concatamer formation of adaptorsequences (see FIG. 6B Panel 3). The resulting adapted workflow in thesequence library construction process when using the modified adaptorapproach is shown schematically in FIG. 6C Panel 4.

The invention claimed is:
 1. A method for identifying one or morepolymorphisms in nucleic acid samples, comprising: (a) performing areproducible complexity reduction on a plurality of nucleic acid samplesto provide a plurality of libraries of the nucleic acid samplescomprising amplified fragments, wherein the reproducible complexityreduction comprises amplifying fragments of the nucleic acid samplesusing one or more primers to obtain the amplified fragments, and whereinthe amplified fragments in each library comprise a unique identifiersequence to indicate origin of each library obtained by the reproduciblecomplexity reduction; (b) combining the plurality of libraries to obtaina combined library and sequencing at least a portion of the combinedlibrary to obtain sequences, wherein the sequencing step (b) isperformed on a solid support; (c) aligning the sequences obtained instep (b) to obtain an alignment; and (d) identifying one or morepolymorphisms in the plurality of nucleic acid samples using thealignment of step (c).
 2. The method according to claim 1, wherein thesolid support is a bead.
 3. The method according to claim 1, wherein thesequencing step (b) comprises dideoxy chain-terminating sequencing. 4.The method according to claim 1, wherein the sequencing step (b)comprises high-throughput sequencing.
 5. The method according to claim2, wherein the sequencing step (b) comprises: (i) annealing amplifiedfragments to beads, each bead annealing with a single amplifiedfragment; (ii) emulsifying the beads in water-in-oil microreactors, eachwater-in-oil microreactor comprising a single bead; (iii) loading thebeads in wells, each well comprising a single bead; and (iv) generatinga pyrophosphate signal.
 6. The method according to claim 5, wherein,preceding the annealing step, sequencing adaptors are ligated to theamplified fragments.
 7. The method according to claim 6, wherein thesequencing adaptors carry a 3′-T overhang.
 8. The method according toclaim 1, wherein at least one of the primers is phosphorylated.
 9. Themethod according to claim 1, wherein the unique identifier sequenceindicates, independently from sequences of the nucleic acid samples,origin of each library obtained by the reproducible complexityreduction.